Biomarkers are cellular, biochemical, or molecular alterations that can be easily and non-invasively measured in human tissues and are directly or indirectly involved in the pathway of disease. Cardiovascular disease (CVD) is the leading cause of death in the United States accounting for 800,000 deaths every year. Both short- and long-term exposure to air pollution/toxic metals has been consistently associated with CVD morbidity and death in epidemiologic studies. Platelets contribute to the development of both atherosclerosis and arterial thrombosis, which are primary contributing events leading to CVD. Increased platelet counts have been suggested to mediate particulate matter (PM) effects. Platelets are anucleate, but mitochondrial DNA (mtDNA) can be easily isolated from plasma platelets. Mitochondria are highly sensitive to PM-related oxidation and, most importantly, platelets are critically dependent on energy production in mitochondria. We hypothesize that air pollutants and metals exposure may cause alteration in platelet mitochondrial DNA methylation (mtDNA) in response to exposure-induced systemic oxidation/inflammation. Platelet mtDNA methylation changes may lead to, or at a minimum reflect mitochondrial dysfunction participating directly or indirectly to the paths linking toxic exposures to CVD risk. We propose t test these hypotheses using data and biospecimens from the Normative Aging Study (NAS), a large prospective cohort of aging individuals. We have modified and adapted protocols to isolate mtDNA from plasma platelets, and successfully tested our highly time- and cost-effective approach. Plasma platelets from 700 participants will be measured at two time points separated by 4 years for mtDNA oxidative damage (8-oxo-dG), mitochondrial global content of both standard DNA methylation (5-mC) and the emerging hydroxymethylation (5-OH-mC) mark, and locus-specific methylation in a comprehensive panel of mtDNA sequences. We will determine the effects of air pollution and metals on mtDNA biomarkers (Aim 1); evaluate further association of mtDNA biomarkers with CVD risk (Aim 2); and conduct an exploratory aim (Aim 3) to determine via mediation analysis whether platelet mtDNA changes are directly or indirectly on the paths linking toxic exposures to oxidation/inflammation markers, and ultimately to CVD risk. The proposed study will be the first to characterize mechanisms in platelet mtDNA methylation related to CVD risk. The project's focus on platelets, a single pure cell population with critical roles in CVD etiology will provide ease of interpretation of epigenetic findings and thus overcome the major limitation of current epigenetic studies in large human population, which have been hampered by the use of mixed cell populations such as white blood cells or buccal cells. Because platelets can be inexpensively and non- invasively isolated from plasma, our study has high potential to generate novel risk and diagnostic biomarkers of future CVD. If we observed the hypothesized associations, we will seek funding to expand this line of research in large-scale studies in diverse populations with high risk of CVD.